DEVELOPMENT AND DISEASE
RESEARCH ARTICLE 2659
Development 135, 2659-2668 (2008) doi:10.1242/dev.019810
Gap junction communication between uterine stromal
cells plays a critical role in pregnancy-associated
neovascularization and embryo survival
Mary J. Laws1, Robert N. Taylor3, Neil Sidell3, Francesco J. DeMayo4, John P. Lydon4, David E. Gutstein5,
Milan K. Bagchi2 and Indrani C. Bagchi1,*
In the uterus, the formation of new maternal blood vessels in the stromal compartment at the time of embryonic implantation is
critical for the establishment and maintenance of pregnancy. Although uterine angiogenesis is known to be influenced by the
steroid hormones estrogen (E) and progesterone (P), the underlying molecular pathways remain poorly understood. Here, we
report that the expression of connexin 43 (Cx43), a major gap junction protein, is markedly enhanced in response to E in uterine
stromal cells surrounding the implanted embryo during the early phases of pregnancy. Conditional deletion of the Cx43 gene in
these stromal cells and the consequent disruption of their gap junctions led to a striking impairment in the development of new
blood vessels within the stromal compartment, resulting in the arrest of embryo growth and early pregnancy loss. Further analysis
of this phenotypical defect revealed that loss of Cx43 expression resulted in aberrant differentiation of uterine stromal cells and
impaired production of several key angiogenic factors, including the vascular endothelial growth factor (Vegf). Ablation of CX43
expression in human endometrial stromal cells in vitro led to similar findings. Collectively, these results uncovered a unique link
between steroid hormone-regulated cell-cell communication within the pregnant uterus and the development of an elaborate
vascular network that supports embryonic growth. Our study presents the first evidence that Cx43-type gap junctions play a critical
and conserved role in modulating stromal differentiation, and regulate the consequent production of crucial paracrine signals that
control uterine neovascularization during implantation.
INTRODUCTION
During the early stages of pregnancy, the steroid hormones estrogen
(E) and progesterone (P) orchestrate the structural and functional
changes in the mammalian uterus that enable the blastocyst to attach
to it, initiating the process of implantation (Psychoyos, 1973;
Yoshinaga, 1988; Parr and Parr, 1989; Weitlauf, 1994). In the mouse
and the human, as the embryo invades into the stromal compartment,
the fibroblastic stromal cells undergo differentiation into a unique
secretory tissue, known as the decidua. This transformation process,
known as decidualization, is crucial for executing the extensive
tissue remodeling that ensures proper maternal-fetal interactions,
leading to the establishment of pregnancy. Decidualization is also
accompanied by the creation of an extensive vascular network
within the stromal bed that supports embryonic and placental growth
and maintains early pregnancy (Cross et al., 1994; Irwin and
Giudice, 1999; Carson et al., 2000).
During angiogenesis, new blood vessels are generated by the
extension of pre-existing vessels into avascular space. This process
involves the local degradation of the vascular basal membrane by
proteases, proliferation and migration of endothelial cells, and
assembly of these cells into new vessels (Folkman, 1995; Hyder and
Stancel, 1999). In the female reproductive system, an active
1
Department of Veterinary Biosciences, and 2Department of Molecular and
Integrative Physiology, University of Illinois Urbana/Champaign, Urbana, IL 61802,
USA. 3Department of Gynecology and Obstetrics, Emory University School of
Medicine, Atlanta, GA 30322, USA. 4Department of Molecular and Cellular Biology,
Baylor College of Medicine, Houston, TX 77030, USA. 5New York University School
of Medicine, NY 10016, USA.
*Author for correspondence (e-mail: ibagchi@uiuc.edu)
Accepted 20 May 2008
angiogenesis is required to support the cyclic remodeling of the
uterus. In the human and the non-human primates, the spiral arteries
that supply the functionalis layer of the endometrium increase in
length, branching and coiling during each menstrual cycle as the
endometrium is regenerated (Hyder and Stancel, 1999). In rodents,
a link between the steroid-driven stromal differentiation program
and active neovascularization within the pregnant uterus has long
been speculated, although the underlying mechanisms are unknown.
One of the earliest signs of a uterine response to an angiogenic
stimulus is an increase in microvascular permeability at the sites of
implantation (Chakraborty et al., 1995; Rockwell et al., 2002). E is
recognized as a regulator of this phenomenon (Chakraborty et al.,
1995; Rockwell et al., 2002). However, the precise nature of the
hormone-regulated pathways that influence uterine angiogenesis
remains unclear and controversial. Particularly intriguing is a
previous report that E is an inhibitor and P is a stimulator of uterine
angiogenesis (Ma et al., 2001). As this study was performed using
ovariectomized non-pregnant mice following treatment with steroid
hormones, the relevance of these findings under normal pregnancy
conditions is questionable. A major challenge in reproductive
medicine is, therefore, to gain a clear understanding of the steroid
hormone-regulated pathways that control pregnancy-associated
endometrial neovascularization in the stromal compartment. The
present study was undertaken to uncover and functionally
characterize these pathways.
MATERIALS AND METHODS
Animals and tissue collection
Mice were maintained in the designated animal care facility at the University
of Illinois College of Veterinary Medicine according to the institutional
guidelines for the care and use of laboratory animals. For mating studies,
DEVELOPMENT
KEY WORDS: Implantation, Endometrium, Neovascularization, Estrogen, Connexin 43, Mouse
Cx43fl/fl and Cx43d/d female mice were housed with wild-type C57BL/6
male mice (Charles Rivers). The presence of a vaginal plug after mating was
designated as day 1 of pregnancy.
In order to induce superovulation, 7- to 8-week-old female mice were
injected intraperitoneally with 5 IU of pregnant mare serum gonadotrophin
(PMSG) and 48 hours later with 5 IU of human chorionic gonadotropin
(hCG). The mice were killed 16-18 hours post-hCG administration, and
oocytes were flushed from the oviducts and counted.
To induce and maintain delayed implantation, mice were ovariectomized
on day 4 (morning) of pregnancy and injected daily with P (2 mg) from days
5-7. To terminate delayed implantation and induce blastocyst attachment,
the P-primed delayed implanting mice were given an injection of E (50 ng)
on the fourth day of the delay (day 8). Mice were killed at different time
points after E injection and uteri were collected.
Decidualization was experimentally induced in non-pregnant mice as
described previously (Cheon et al., 2004). Mice were first ovariectomized.
Two weeks following ovariectomy, animals were injected with 100 ng of E
in 0.1 ml of sesame oil for 3 consecutive days. This was followed by daily
injections of 1 mg of P for 3 consecutive days. Decidualization was then
initiated in one horn by injection of 50 μl oil. The other horn was left
unstimulated. The animals were treated with P for an additional 6 days poststimulation and then killed to collect the uterine tissue.
Immunohistochemistry
For Cx43, Pecam, Ki67 and Vegf immunostaining, uterine sections were
obtained and flash frozen. Samples were embedded in OCT, cryosections
were taken at 8 μm and subjected to immunostaining with antibodies against
Cx43 (Zymed), Pecam1 (BD Biosciences), Ki67 (Santa Cruz Biotech) and
Vegf (Santa Cruz Biotech). For double immunostaining against Pecam1 and
Cx43, uterine tissues were collected from mice on day 8 of pregnancy and
flash frozen in liquid nitrogen. Incubations with primary antibody were
carried out overnight at 4°C (1:1000 dilution of rat anti-Pecam1 antibody,
Pharmingen 557355; 1:500 dilution of a rabbit polyclonal antibody against
Cx43, Zymed 71-0700), using frozen sections. Secondary antibody
incubations were carried out for 2 hours at room temperature with a 1:200
dilution of fluorescently conjugated secondary antibodies: TRITCconjugated goat anti-rabbit antibody (Sigma T6778) and FITC-conjugated
goat anti-rat antibody (Sigma F6258). Sections were washed three times for
5 minutes each in PBS.
Human endometrial stromal cells
Primary human endometrial stromal cells were isolated from endometrial
biopsies of fertile women and immortalized by stable transfection of a gene
coding for an essential catalytic protein subunit of human telomerase
reverse transcriptase (Krikun et al., 2004). These telomerase-expressing
stromal cells (termed HESC-T) were stably transfected with retroviral
vectors expressing Cx43 small interfering RNA (siRNA) and a control
Cx43 non-silencing sequence (Shao et al., 2005). The siRNA insertcontaining retroviral vectors were first transduced into PT67 retropackaging cells (BD Biosciences) to generate infectious viral particlecontaining supernatant. Filtered supernatants were then used to infect
HESC-T cells and the infected cells selected in media containing 50 μg/ml
hygromycin. Cells transfected with a control non-silencing siRNA and
Cx43 siRNA are designated as HESC-TC and HESC-T3, respectively. The
cells were grown in DMEM/F-12 medium containing 5% charcoal-stripped
FBS. To induce in vitro decidualization, the cells were treated with or
without a hormone cocktail containing 1 nM E, 1 mM P and 0.5 mM 8bromo-cAMP for 7-11 days. Cell culture supernatants were collected, and
prolactin and VEGF were measured using standardized ELISA kits. Three
independent experiments were performed to assure reproducibility and the
data are presented as mean±s.d. Comparisons between HESC-TC and
HESC-T3 cells were made using two-tailed Student’s t-tests, with a
significance threshold set at P=0.05.
Dual label cell coupling assay
Donor cells were double labeled with the fluorescent dyes calcein (gap
junction permeable dye) and Dil (gap junction impermeable dye). These
cells were then placed in contact with unloaded cells in a monolayer. Dye
transfer was visualized after 2 hours. The cells that fluoresce both green
Development 135 (15)
(calcein) and red (Dil) are the dual-loaded donor cells, whereas those
fluorescing only green were originally unlabeled in the monolayer and now
demonstrate functional coupling.
RESULTS
To identify steroid-regulated gene networks with functional
relevance in implantation, we used a delayed implantation mouse
model in which implantation is induced by an acute administration
of E to ovariectomized pregnant mice maintained in the presence of
P (Yoshinaga and Adams, 1966; Gidley-Baird, 1981). This
hormonal profile mimics the transient preimplantation surge of E
that is essential for implantation in the rodents. Gene expression
profiling using Affymetrix mouse microarrays (430 2.0 Array)
revealed that connexin 43 (Cx43), also known as gap junction
protein alpha 1 (Gja1), is one of the many genes whose expression
is altered in P-primed uteri in response to E (Mantena et al., 2006)
(M.K.B. and I.C.B., unpublished). The connexins are a family of
transmembrane proteins that form hexameric assemblies in the
plasma membrane to create gap junctions, regulating intercellular
communication. Cx43 is the principal and most well-studied
component of the gap junctions (Kumar and Gilula, 1996; Bruzzone
et al., 1996).
We first confirmed the hormonal regulation of Cx43 expression
in pregnant uterus by performing immunohistochemical analysis.
We observed that E treatment dramatically enhances the expression
of Cx43 protein in the uterine stromal compartment during delayed
implantation (Fig. 1A). Our findings are also in agreement with
previous reports that Cx43 expression in the uterus is primarily
under E regulation (Grummer et al., 2004). When we examined the
profile of Cx43 protein expression in mouse uterus during normal
pregnancy, it was undetectable in undifferentiated stromal cells
during the preimplantation period (Fig. 1B, parts a,b). However, on
day 5 of pregnancy, within 12 hours of the initiation of implantation,
a marked induction in Cx43 protein expression was observed in the
stromal cells in the primary decidual zone immediately surrounding
the implanting embryo (Fig. 1B, parts c,d). As pregnancy progressed
to day 7, Cx43 expression intensified and spread to the secondary
decidual zone (Fig. 1B, parts e,f). The close spatio-temporal
relationship between Cx43 expression and the progression of
decidualization raised the possibility that stromal gap junctions
harboring this protein may play an important role during the
differentiation process.
To investigate the function of Cx43 during embryo implantation,
we employed a loss-of-function approach using genetically
engineered mice. Cx43-null mice exhibit a perinatal lethal
phenotype due to impaired cardiovascular development (Reaume et
al., 1995). To circumvent this problem, we created a conditional
knockout of the Cx43 gene in the uterus of adult mice by employing
the Cre-LoxP strategy. Transgenic mice expressing Cre under the
control of progesterone receptor (PR) promoter were used
previously to ablate ‘floxed’ genes in the uterus (Lee et al., 2006;
Mukherjee et al., 2006; Lee et al., 2007). We crossed these PR-Cre
mice with those harboring the ‘floxed’ Cx43 gene (Cx43fl/fl) to create
the Cx43d/d mice in which the Cx43 gene is deleted in uterine cells
expressing PR. The ablation of the Cx43 gene in the uterine tissue
of Cx43d/d mice during pregnancy was confirmed when uterine
sections obtained from these mice failed to show any Cx43 protein
expression in the stromal cells surrounding the implanted embryo
(Fig. 2).
An 8-month breeding study demonstrated that female Cx43d/d
mice exhibit severe fertility defects (Table 1). Our study revealed
that ~50% of a cohort (n=13) of Cx43d/d female mice analyzed
DEVELOPMENT
2660 RESEARCH ARTICLE
Role of connexin 43 in implantation
RESEARCH ARTICLE 2661
Fig. 1. Induction of Cx43 expression in uterine stromal cells
during implantation. (A) Mice were subjected to delayed
implantation as described in the Materials and methods. Uteri were
collected 1 hour (b) and 24 hours (c,d) after E treatment and subjected
to immunohistochemistry using anti-Cx43 antibody. (d) Higher
magnification view of the 24-hour sample. The 0-hour sample (a)
represents pregnant uterus obtained from a mouse treated with P alone
for three days. Results are from two independent experiments. (B) Uteri
were collected in the morning of day 1 (a), day 4 (b), day 5 (c,d) and
day 7 (e,f) of pregnancy. Higher magnifications of Cx43 expression in
stromal cells surrounding the implanted embryo on day 5 and day 7 are
shown in d and f, respectively. Results are from two independent
experiments.
during this breeding experiment never gave birth, although they
mated normally with wild-type males. Furthermore, the Cx43d/d
mice that did give birth exhibited a more than 60% reduction in the
number of pups per litter when compared with control Cx43fl/fl mice.
Overall, these results indicated that the conditional excision of the
Cx43 gene led to an ~80% reduction in the total number of pups born
per Cx43d/d female compared with a Cx43fl/fl female in the breeding
program (35/13 versus 419/37). Superovulation experiments
indicated that Cx43d/d mice ovulate normally and release oocytes in
quantities comparable to those of Cx43fl/fl mice (Table 1). In
agreement with normal ovarian activity, serum P levels were normal
in the Cx43d/d mice on day 8 of pregnancy (Table 1). Collectively,
these data suggest that the observed fertility defect in Cx43d/d mice
is not likely to be due to an impairment of the hypothalamicpituitary-ovarian axis.
Further analysis indicated that the Cx43d/d mice are able to initiate
embryo implantation and support pregnancy up to day 7 of gestation.
Implanted embryos embedded in the stroma were observed in both
Cx43fl/fl and Cx43d/d mice (Fig. 3A, parts a,c). However, starting on
day 8 of pregnancy in Cx43d/d mice, we detected distinct signs of
arrest of embryonic growth and noted embryo loss or resorption.
Morphological analysis of uterine sections obtained from these mice
on day 8 of pregnancy showed abnormally small embryos compared
with those of Cx43fl/fl mice (compare parts b and d in Fig. 3A). An
examination of the histological sections from day 8 pregnant uteri
revealed a severe impairment in the development of an angiogenic
network in the stromal bed of Cx43-deficient uteri (Fig. 3A, parts
e,f). When the uterine sections of pregnant Cx43fl/fl mice were
subjected to immunohistochemistry using an antibody against
platelet/endothelial cell adhesion molecule (Pecam), a marker of
endothelial cells, they displayed a well-developed vascular network
spanning the endometrial bed that surrounds the implanted embryo
on day 7 or day 8 of pregnancy (Fig. 3B, parts a-c). The Pecam
immunostaining, however, was reduced drastically in uterine
sections of Cx43d/d mice on day 7 or day 8 of pregnancy, indicating
that only a rudimentary vasculature formed in the mutant uteri
(Fig. 3B, parts d-f). It is important to mention here that
immunofluorescence experiments using differently labeled
antibodies against Pecam and Cx43 indicated that during early
pregnancy Cx43 expression occurs solely in the uterine stromal cells
and not in the endothelial cells (Fig. 3C). These results support the
concept that the loss of Cx43 expression in the stromal gap junctions
is responsible for the drastic reduction in the endothelial cell
population in the pregnant uterus.
The lack of endothelial cell proliferation in Cx43-deficient uteri
was further ascertained by immunostaining for Ki67, a marker for
cell proliferation (Fig. 3D). Whereas the uterine sections obtained
from Cx43fl/fl mice on day 8 of pregnancy exhibited robust Ki67
immunostaining in endothelial cells, consistent with microvascular
DEVELOPMENT
Fig. 2. Loss of Cx43 expression in the uterus of the Cx43
conditional-knockout mouse. (A,B) Uterine sections from Cx43fl/fl
(control) mice show prominent Cx43 expression in the stromal cells
surrounding the embryo. (C,D) Uterine sections from Cx43d/d
(conditional knockout) mice show efficient ablation of Cx43 in uterine
stromal cells. E denotes implanted embryo.
2662 RESEARCH ARTICLE
Development 135 (15)
Table 1. Impact of Cx43 ablation on female fertility
Genotype
Cx43fl/fl
Cx43d/d
Number
Number
of pups
Number
of litters
Average
pups/litter
Ovulation
(ova/ovary)*
Serum P4
(ng/ml)*
37
7†
419
35
65
13
6.4
2.6
19±2
15±1
84±12
95±3
Serum P concentrations were measured by radioimmunoassay (RIA) at the core facility of University of Virginia at Charlottesville. The values are expressed as mean±s.e.m.
*n=5.
†
Thirteen female mice were placed in the breeding program; six mice did not give birth.
analyzed the decidual response in Cx43d/d uteri by monitoring the
expression of decidual prolactin-related protein (PRP) and prolactinlike protein (PLP), well-known biochemical markers of
decidualization (Orwig et al., 1997; Rasmussen et al., 1997; Croze
et al., 1990; Lin et al., 1997). As expected, uterine sections from
Cx43fl/fl mice exhibited prominent PRP expression in both
antimesometrial (AM) and mesometrial (M) areas on day 7 of
Fig. 3. Impaired embryo development and lack of angiogenesis in uteri of Cx43 conditional knockout mice on day 8 of gestation.
(A) Hematoxylin and Eosin staining of uterine sections obtained from Cx43fl/fl (a,b) and Cx43d/d (c,d) mice on days 7 and 8 of pregnancy. Note that
implanted embryos (E) develop poorly in Cx43d/d mice compared with those in Cx43fl/fl mice. Histological analysis by Hematoxylin and Eosin staining
of perfused uterine sections obtained from pregnant Cx43fl/fl (e) and Cx43d/d (f) mice on day 8 of pregnancy. E, implanted embryo; M, the
mesometrial region. Note the extensive vasculature in the uteri of Cx43fl/fl mice and the lack of it in the uteri of Cx43d/d mice.
(B) Immunohistochemical staining (indicated by red deposits) for Pecam in pregnant uteri. Uterine sections of Cx43fl/fl (a-c) and Cx43d/d (d-f) mice on
day 7 (a,b,d,e) and day 8 (c,f) of pregnancy are shown (n=3). (C) Uterine sections collected from mice on day 8 of pregnancy were subjected to
immunoflouroscence employing anti-connexin 43 (a) and anti-Pecam1 (b) antibodies. c represents a merge of both Cx43 and Pecam staining
indicating the expression of Cx43 in the stromal cells but not in the endothelial cells (n=2). (D) Uterine sections from Cx43fl/fl (a) and Cx43d/d (b)
mice on day 8 of gestation were subjected to immunohistochemistry using an antibody against Ki67, a marker of cell proliferation (n=2).
DEVELOPMENT
proliferation (Fig. 3D, part a), those obtained from mutant animals
showed greatly reduced Ki67 staining (Fig. 3D, part b), confirming
a severely compromised endothelial cell proliferation in the absence
of stromal Cx43 expression.
To gain further insight into the mechanisms underlying the
phenotypical defects of Cx43d/d uteri, we examined whether the loss
of Cx43 expression affected the stromal differentiation program. We
pregnancy (Fig. 4A). A similar pattern of PRP expression was
observed in Cx43-deficient pregnant uterus on this day (Fig. 4B). On
day 8 of pregnancy in Cx43fl/fl mice, PRP expression was drastically
reduced in the decidual cells at both antimesometrial and
mesometrial regions. Only a few cells in the mesometrial region
close to the ectoplacental cone retained strong PRP expression (Fig.
4C). By contrast, a strikingly different spatial expression of PRP was
seen in Cx43-deficient uteri on day 8 of pregnancy (Fig. 4D). In
these mutant uteri, expression of PRP, similar to that seen on day 7
of pregnancy, persisted in the majority of the decidual cells in the
antimesometrial and mesometrial areas. A plausible explanation for
the absence of timely downregulation of PRP expression in these
cells is that they fail to reach a more advanced state of
differentiation. When we analyzed PLP expression, it was prominent
in the antimesometrial region of uterine sections of Cx43fl/fl mice on
day 8 of pregnancy and was markedly reduced in Cx43-deficient
uteri (Fig. 4E,F). Collectively, the aberrant expression of both PRP
and PLP indicated that the loss of Cx43 expression in the uterine
stromal cells leads to an impairment in the proper progression of the
decidualization program.
It is important to address whether Cx43 controls stromal
decidualization and angiogenesis independently of embryonic
development. We, therefore, subjected non-pregnant mice to
experimentally induced decidualization in which a mechanical
perturbation of the steroid-primed uteri triggers a decidual response
in the absence of the implanting embryo (Cheon et al., 2004). Uteri
of ovariectomized Cx43fl/fl and Cx43d/d mice were prepared by
treating these animals with a well-established regimen of E and P, and
then decidualization was initiated in the left uterine horn by injecting
Fig. 4. Cx43-deficient uteri exhibit impaired decidualization.
(A-D) Uterine sections from Cx43fl/fl and Cx43d/d mice on day 7 (A,B)
and day 8 (C,D) of pregnancy were subjected to immunohistochemical
staining using an antibody specific for the prolactin-related protein
(PRP). (E,F) Uterine sections from Cx43fl/fl (E) and Cx43d/d (F) mice on
day 8 of pregnancy were subjected to immunohistochemical staining
using an antibody specific for the prolactin-like protein (PLP). AM and
M denote antimesometrial and mesometrial regions, respectively.
Results are from two independent experiments.
RESEARCH ARTICLE 2663
50 μl oil while the right horn was left unstimulated. As shown in Fig.
5A, widespread expression of Cx43 is induced in stromal cells of
Cx43fl/fl mice in response to the deciduogenic stimulus. We then
examined the gross anatomy of the stimulated and unstimulated
uterine horns of Cx43fl/fl and Cx43d/d mice. As expected, the uterine
horn of Cx43fl/fl mice exhibited a robust decidual response within 48
hours of receiving the artificial stimulation (Fig. 5B, upper left panel).
By contrast, the Cx43-deficient uteri under identical conditions
showed significantly reduced decidualization (Fig. 5B, upper right
panel). When the decidual response was assessed by measurement of
uterine wet weight gain, the Cx43-deficient uteri exhibited a
markedly reduced weight gain relative to that seen in the Cx43fl/fl
uteri (Fig. 5B, middle panel). We further analyzed the decidualization
response of Cx43d/d uteri by monitoring the expression of Hoxa10
and bone morphogenetic protein 2 (Bmp2), factors that are induced
in stromal cells during decidualization and that play important
regulatory roles during this process (Lim et al., 1999; Lee et al., 2007;
Li et al., 2007). As shown in the lower panel of Fig. 5B, when
Cx43fl/fl and Cx43-deficient uteri were subjected to artificial decidual
stimulation, we observed a marked downregulation of mRNAs
corresponding to Hoxa10 and Bmp2 in the uteri lacking Cx43,
consistent with the other decidualization defects observed in the
conditional mutant mice.
We next analyzed the angiogenic capacity of Cx43-deficient uteri
during artificial decidualization. Immunohistochemical analysis of
Cx43fl/fl and Cx43-deficient uteri with a Pecam antibody revealed
that the artificially stimulated Cx43fl/fl horn displayed an extensive
endothelial cell network spanning the endometrial bed (Fig. 5C, left
panel). By contrast, similarly treated uterine horns of Cx43d/d mice
displayed drastically reduced Pecam staining, indicating that only a
rudimentary vasculature is formed (Fig. 5C, right panel). These
studies clearly indicate that even in the absence of the conceptus,
communication via Cx43 gap junctions plays a crucial role in
stromal cell differentiation and angiogenesis in the steroid hormoneprimed uterus.
We considered the possibility that the impaired decidualization of
the mutant stromal cells might affect the timely production of
paracrine regulators from these cells, thereby inhibiting endothelial
proliferation or angiogenesis. Ample evidence indicates that Vegf is
a potent paracrine stimulator of endothelial cell proliferation and is
a crucial angiogenic factor during decidualization (Ferrara et al.,
1996; Halder et al., 2000). We, therefore, examined the pattern of
Vegf protein expression in Cx43fl/fl and Cx43d/d uteri during the
decidualization phase. Widespread expression of Vegf was observed
in Cx43fl/fl uteri on days 7 (Fig. 6A, part a) and 8 (Fig. 6A, part b) of
pregnancy, particularly in the mesometrial area, which is the primary
source of the growing implantation site vasculature. Its spatial
expression pattern closely overlapped with that of Pecam, which
marked the endothelial network (compare Fig. 3B and Fig. 6A). By
contrast, a significant downregulation of Vegf expression,
concomitant with the sharp reduction in Pecam immunostaining,
was seen in Cx43-deficient uteri (Fig. 6A, part c,d; compare with
Fig. 3B, parts d-f). The Vegf expression in Cx43d/d uteri was limited
to only a few layers of cells surrounding the implanted embryo, and
was markedly reduced in the mesometrial region. We also observed
a marked downregulation of mRNAs encoding angiopoietin 2 and
angiopoietin 4 in Cx43-deficient uteri during the decidualization
phase (Fig. 6B). As these factors are known to play important
regulatory roles in endothelial cell proliferation, migration and new
blood vessel formation, our findings provide mechanistic insights
into the pathways via which stromal Cx43 gap junctions control
angiogenesis during decidualization.
DEVELOPMENT
Role of connexin 43 in implantation
2664 RESEARCH ARTICLE
Development 135 (15)
To further explore the relationship between Cx43 and Vegf
expression, and to test whether this important functional link is
conserved among the species, we extended our study to human
endometrial stromal cells, which are known to produce these
proteins during decidualization (Jahn et al., 1995; Shifren et al.,
1996; Granot et al., 2000). We used primary human endometrial
stromal cells (HESC-T) that have been immortalized by stable
transfection of a gene encoding the catalytic subunit of human
telomerase (Krikun et al., 2004). By using these cells, we
established a low CX43-expressing human stromal cell line HESCT3 that is stably transduced with a retroviral vector expressing
small interfering RNA (siRNA) targeted to CX43 mRNA,
generously provided by Dr Dale Laird (Shao et al., 2005). A control
cell line, HESC-TC, containing the same retroviral vector
expressing a non-target sequence was also generated.
Quantification by real-time RT-PCR indicated that CX43 mRNA
levels in HESC-T3 cells were drastically reduced (>90%) relative
to those in control HESC-TC cells (data not shown).
Correspondingly, western blot analysis demonstrated a marked
reduction of CX43 protein in HESC-T3 cells (Fig. 7A). To examine
whether the consequence of this forced suppression of CX43 is an
inhibition of gap junctions, we used a double dye-labeling
technique. As shown in Fig. 7B, gap junction-permeable green
calcein dye diffused from injected control HESC-TC cells (yellow
arrows, Fig. 7B) to adjacent cells, confirming that functional gap
junctions exist between stromal cells. By contrast, the injected dye
failed to diffuse from low CX43-expressing HESC-T3 stromal
cells. The non-diffusible red DiI marker identifies the microinjected
cells. Interestingly, the HESC-T3 cells also failed to undergo
morphological decidualization in vitro following treatment with a
hormonal cocktail containing E, P and cAMP, whereas the control
HESC-TC cells, treated with this cocktail, exhibited distinct
epithelioid morphological characteristics (Ryan et al., 1994) that
were indicative of their differentiated status (Fig. 7C). These results
correlate with the impaired progression through decidualization
displayed by the uterine stromal cells of Cx43d/d mice.
We next examined whether this lack of stromal differentiation in
the absence of CX43 resulted in differences in protein production
from HESC-TC and HECS-T3 cells. Prolactin, a biomarker of
decidualization in human endometrial stromal cells, similar to the
expression of PRP in the mouse, was undetectable in untreated cells
but was induced after 7-11 days incubation with E, P and cAMP.
Prolactin production in hormone-treated HESC-T3 cells was
reduced by 66±8% relative to control HESC-TC cells (n=3, P<0.02).
DEVELOPMENT
Fig. 5. Cx43 is essential for decidual response and angiogenesis.
(A) Expression of Cx43 in artificially decidualized uterus. Mice were
subjected to artificial decidual stimulation. Uteri were collected at 0 (a)
and 72 (b,c) hours following the application of the stimulus. Uterine
sections were analyzed for Cx43 expression by immunohistochemistry.
c is a higher magnification of the 72-hour sample. (B, upper) Gross
morphology. Cx43fl/fl (left) and Cx43d/d (right) mice were subjected to
artificial decidual stimulation for 48 hours as described in the Materials
and methods. For each mouse, one uterine horn was stimulated, while
the other horn was left undisturbed. The stimulated horn is indicated as
‘s’ and the unstimulated one as ‘us’. (Middle) Comparative wet weight
gains in uteri of Cx43fl/fl and Cx43d/d mice. Following artificial
decidualization, stimulated and unstimulated horns were assessed for
wet weight gain. The histogram shows the ratios of average weights of
stimulated over unstimulated horns from Cx43fl/fl and Cx43d/d mice. The
data are represented as means±s.e.m. (n=4). (Lower) Expression of
Hoxa10 and Bmp2 mRNA in the uteri of Cx43fl/fl and Cx43d/d mice.
Total RNA was isolated from uteri collected 96 hours after the
application of the artificial decidual stimulus and qPCR analysis was
performed using gene-specific primers. (C) Immunohistochemical
staining for Pecam in artificially decidualized uteri. Uterine sections of
Cx43fl/fl (a) and Cx43d/d (b) mice are shown. The results are
representative of two independent experiments.
Fig. 6. Reduced expression of angiogenic factors in Cx43deficient uteri. (A) Uterine sections from Cx43fl/fl mice on day 7 (a)
and day 8 (b) and those from Cx43d/d mice on day 7 (c) and day 8 (d)
were subjected to immunohistochemical analysis using anti-Vegf
antibody. E, implanted embryo; M, the mesometrial region. (B) qPCR
analysis was performed to monitor angiopoetin 2 and angiopoetin 4
mRNA expression in the uteri of Cx43fl/fl and Cx43d/d mice on day 8 of
pregnancy (n=3).
In addition to a reduction in this classical marker of stromal
differentiation, Fig. 7D indicates that basal and phorbol ester (TPA)induced production of VEGF also was reduced in low CX43
expressing HESC-T3 cells compared with control HESC-TC cells.
Quantification of VEGF synthesis was performed in three
independent E, P and cAMP-treated cell cultures. In control HESCTC cells, hormone treatment stimulated VEGF production 11.4±5.7
fold, whereas the hormone-induced augmentation in HESC-T3 cells
was only 1.4±0.1-fold (n=3, P<0.04). Our findings indicate that
CX43 gap junctions are directly involved in the regulation of VEGF
production in human endometrial stromal cells during in vitro
decidualization. Furthermore, conservation of the important
functional link between CX43 and VEGF expression in both mouse
and human stromal cells supports a fundamental role of gap junction
communication during uterine angiogenesis.
DISCUSSION
In rodents, a transient surge of E in the preimplantation phase is
essential for initiating implantation of the blastocyst into the uterine
epithelium (Psychoyos, 1973; Yoshinaga, 1988). The E-induced
delayed implantation in rats and mice faithfully captures this
hormonal effect. Although the implantation-inducing action of E in
rodents has been known for a long time, the mechanism and
mediators of this effect remain largely unknown. Using gene
RESEARCH ARTICLE 2665
expression profiling, we have identified Cx43 as a target of E
regulation in the uterus during delayed implantation. The connexins
constitute a large family of gap junction proteins that regulate
intercellular communications. Although other members of this
family, including Cx26 and Cx31, are expressed in the pregnant
mouse uterus (Grummer et al., 1996), our microarray data indicated
that Cx43 is the sole member of this family whose uterine expression
is induced by E during implantation. Our findings are in agreement
with previous reports that Cx43 expression in the uterus is primarily
under E regulation (Grummer et al., 2004).
The expression of Cx43 in uterine stromal cells is intimately
associated with the decidualization phase of pregnancy.
Following blastocyst attachment, the underlying stromal cells
undergo extensive proliferation and differentiation that result in
their transformation into the decidual cells. In mice and a few
other species, decidualization can also be experimentally induced
by a variety of artificial stimuli in steroid hormone-primed uteri.
We found that the expression of Cx43 is robustly induced in the
decidual tissue during both normal and artificial decidualization.
This induction of Cx43 is likely to be regulated by E acting via
ERα (Esr1 – Mouse Genome Informatics). In support of this view,
our recent studies using conditional knockout mice harboring a
null mutation of the gene encoding ERα in the uterus have shown
that E-induced expression of Cx43 is absent in these mutant mice
(M.J.L. and I.C.B., unpublished). A direct regulatory role for ERα
in Cx43 expression, however, remains to be established, and
would require a detailed analysis of the 5⬘-flanking regulatory
region of the Cx43 gene for the presence of functional ER-binding
sites.
Analysis of the Cx43-deficient uteri revealed an impaired
decidual response during both normal and artificial decidual
reaction. A defect in decidualization could arise from either
compromised stromal cell proliferation or an arrest in the
differentiation program of these cells. Immunohistochemical
studies using the cell proliferation marker Ki67 indicated that
Cx43-deficiency did not significantly affect stromal cell
proliferation, which occurs predominantly during days 5 and 6 of
pregnancy (data not shown). However, in the absence of Cx43 gap
junctions, the stromal cells failed to progress properly in the
differentiation program initiated in response to a decidual
stimulation. This was evidenced by a marked reduction in uterine
wet weight gain, which is considered a hallmark of
decidualization. Additionally, two well-known markers of uterine
stromal differentiation, PRP and PLP, exhibited impaired or
aberrant expression in Cx43-deficient uteri during days 7 and 8 of
pregnancy. The altered expression of these biomarkers indicated
that the loss of Cx43 expression in the uterine stromal cells leads
to improper progression of the decidualization program. The
defect in stromal differentiation in the absence of Cx43 was further
substantiated when we analyzed the uterine expression of Hoxa10
and Bmp2, which are crucial regulators of decidualization.
Previous studies have shown that mice harboring a targeted
deletion of either the Hoxa10 or the Bmp2 gene exhibit impaired
uterine stromal differentiation and are infertile (Lim et al., 1999;
Lee et al., 2007). The expression of both of these factors was
markedly reduced in Cx43-deficient uteri. Furthermore, we
observed that the loss of Cx43 expression in human endometrial
stromal cells blocked their differentiation into prolactin-producing
decidual cells. Collectively, these results form the basis of the
important concept that the formation of Cx43 gap junctions
between stromal cells is critical for the efficient and timely
progression of the decidualization program.
DEVELOPMENT
Role of connexin 43 in implantation
2666 RESEARCH ARTICLE
Development 135 (15)
Our study also suggests that stromal differentiation and
angiogenesis are intimately linked processes within the pregnant
uterus. Paracrine factors secreted by the decidualizing stromal cells
might influence the proliferation and function of uterine endothelial
cells in the mesometrial region of the pregnant uterus where
neovascularization mostly occurs. Impaired decidualization due to
the loss of Cx43 gap junction communication might result in
reduced expression and secretion of angiogenic factors by the
uterine stromal cells, and might thereby lead to a concomitant
impairment in angiogenesis. In strong support of this concept, we
observed a markedly reduced expression of Vegf, a potent stimulator
of endothelial cell proliferation and a well-known angiogenic
molecule, particularly in the mesometrial region of the pregnant
Cx43-deficient uterus. Previous studies have shown that the
morphogen Bmp2 mediates the enhancement of Vegf expression and
neovascularization in xenografts of breast tumors in mice (Raida et
al., 2005). It was also demonstrated that Vegf expression in
chondrocytes requires cooperative interactions between the Bmp2
and β-catenin signaling pathways (Chen et al., 2008). It is, therefore,
conceivable that Bmp2 produced by the decidualizing stromal cells
might regulate uterine Vegf expression during early pregnancy.
Future studies will evaluate the role of Bmp2 and other stromaderived factors in the regulation Vegf expression in the decidual
uterus.
We also noted that the expression of two additional angiogenesis
regulators, angiopoietins 2 and 4, was downregulated in the Cx43deficient uterus during the decidualization phase. Whereas previous
studies established that angiopoietin 2 is required for the sprouting
of new blood vessels in adult tissues, recent reports implicated
angiopoietin 4 in endothelial cell migration (Gale et al., 2002; Lee
et al., 2004). The lack of expression of these crucial angiogenic
factors was predictably associated with a dramatic impairment in the
formation of neovasculature in pregnant Cx43-deficient uteri, and,
consequently, led to poor embryo development and early pregnancy
loss. Most importantly, the use of primary cultures of human
endometrial stromal cells allowed us to establish that the
DEVELOPMENT
Fig. 7. Attenuation of CX43 expression in human endometrial stromal cells leads to impaired gap junction communication,
decidualization and a reduction in VEGF production. (A) Western blotting was used to analyze the expression of CX43 in HESC-T cells stably
transfected with a CX43 siRNA (HESC-T3) or a nonsilencing control vector (HESC-TC). Pronounced silencing of CX43 protein was observed in HESCT3 cells, whereas the levels of beta actin protein remained unaltered. (B) Gap junction intercellular communication was determined using a doublelabeling technique. Donor cells were double labeled with the fluorescent dyes calcein (gap junction permeable dye, green fluorescence) and DiI (gap
junction impermeable dye, red fluorescence), and were then placed in contact with unloaded cells in the monolayer. Dye transfer was visualized
after 2 hours. HESC-T3 cells with reduced CX43 expression exhibited lack of green dye diffusion. Arrows show examples of functional coupling
between HESC-TC cells. (C) HESC-TC and HESC-T3 cells were grown in DMEM/F-12 medium containing 5% charcoal-stripped FBS. To induce in
vitro decidualization, they were treated with or without a hormone cocktail containing 1 nM E, 1 mM P and 0.5 mM 8-bromo-cAMP for 7-11 days
(Ryan et al., 1994). When the cells were examined morphologically, a distinct transition from fibroblastic to a plump, epitheloid phenotype,
characteristic of decidual cells, was observed in control HESC-TC cells. Low CX43-expressing HESC-T3 cells failed to show this morphological
transformation. (D) HESC-TC and HESC-T3 cells were grown for 24 hours in the absence (Con) or presence (TPA) of 50 nM phorbol ester. The VEGF
in supernatant was measured in duplicate by ELISA.
interrelationship between stromal CX43 expression, the progression
of decidualization, and the production of VEGF by the decidual cells
also exists in the human. Our study, therefore, uncovered a
conserved, fundamental role of Cx43 gap junctions in maintaining
the stromal differentiation program and supporting angiogenesis in
the uterus during early pregnancy. Another tantalizing possibility is
that the Cx43 gap junctions additionally play a role in the regulation
of communication between the maternal decidua and the conceptus.
Further studies will be required to carefully examine whether Cx43
expression is linked to proper functioning of the extraembryonic
tissues, such as the parietal endoderm, giant cells and the
ectoplacental cone.
Gap junction channels allow direct exchange of ions, second
messengers, metabolites, and other small molecules (up to ~1200
Da) between the cytoplasms of adjacent cells. This type of
intercellular coupling is known to regulate cell proliferation and
differentiation (Lo and Gilula, 1979; Simon and Goodenough,
1998; Evans and Martin, 2002). The identity of the intercellular
signal(s) being exchanged via the stromal gap junction during
decidualization needs to be established. Within the pregnant
uterus, stromal differentiation proceeds in a strictly coordinated
manner, spreading spatially from the antimesometrial to the
mesometrial side as the pregnancy advances. It is conceivable that
stromal cells that are more advanced in the differentiation program
could facilitate the progression of their less differentiated
neighbors by establishing direct contact with these cells. Once the
gap junctions are formed, crucial signaling molecules may pass
from the donor to the recipient cells via these connections. These
regulatory molecules, which may include second messengers, such
as cyclic nucleotides, calcium ions or prostaglandins, are likely to
impact the gene expression program and differentiation fates of the
recipient stromal cells, altering their ability to produce Vegf,
angiopoetin 2, angiopoetin 4, and possibly other paracrine
angiogenic effectors. In support of this concept, cAMP and
phorbol ester-dependent signaling pathways were found to
enhance VEGF expression by human endometrial stromal cells
(Popovici et al., 1999; Fig. 7D). cAMP was also implicated as the
signaling molecule that passes through the gap junctions to
influence the adrenocorticotropin-stimulated growth and
steroidogenic function of adrenal cells (Murray and Fletcher, 1984;
Shah and Murray, 2001). We postulate that a similar mode of
intercellular coupling via Cx43 gap junctions may underlie the
unique spatiotemporal appearance of the differentiation markers
and the coordinated production of angiogenic regulators in the
uterus during early pregnancy.
In summary, the present study revealed a central role for Cx43containing stromal gap junctions in the establishment of an
elaborate vascular network within the endometrial bed that is
essential for successful implantation and subsequent embryonic
growth. The ovarian hormone dependence of Cx43 functionally
regulates gap junction communication among stromal cells,
advancing the process of decidualization and stimulating these cells
to produce crucial paracrine effectors, such as Vegf. Most
importantly, a novel, linear molecular pathway has emerged
providing yet another link between nuclear actions of steroid
hormones and cellular signaling at the level of the plasma
membrane. Ultimately, these molecules control stromal
differentiation and angiogenesis during early pregnancy.
This work was supported by the NICHD/NIH, through cooperative
agreement U54 HD055787, as part of the Specialized Cooperative Centers
Program in Reproduction and Infertility Research. This work was also
supported by National Institutes of Health Grants R01 HD-43381 (I.C.B.),
RESEARCH ARTICLE 2667
R01 HD-44611 (M.K.B.), R01 HD-33238 (R.N.T.) and R01 HD-55379 (N.S.).
This investigation was conducted in a facility constructed with support from
Research Facilities Improvement Program Grant Number C06 RR16515-01
from the National Center for Research Resources, National Institutes of
Health (I.C.B.).
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